Cells in multicellular organisms recognize neighboring cells, adhere to each other and form intercellular junctions. Such junctions play essential roles in various cellular functions, including morphogenesis, differentiation, proliferation and migration (see M. Takeichi, “Cadherin cell adhesion receptors as a morphogenetic regulator.” Science 1991, 251: 1451-1455; B. M. Gumbiner, “Cell adhesion: the molecular basis of tissue architecture and morphogenesis.” Cell 1996, 84: 345-357; K. Vleminckx and R. Kemler, “Cadherins and tissue formation: integrating adhesion and signaling.” Bioassays 1999, 21: 211-220; U. Tepass et al., “Cadherins in embryonic and neural morphogenesis.” Nat. Rev. Mol. Cell Biol. 2000, 1: 91-100; M. Takeichi et al., “Patterning of cell assemblies regulated by adhesion receptors of the cadherin superfamily.” Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2000, 355: 885-890; T. Yagi and M. Takeichi, “Cadherin superfamily genes: functions, genomic organization, and neurologic diversity.” Genes Dev. 2000, 14: 1169-1180). In polarized epithelial cells, intercellular adhesion is mediated through a junctional complex composed of tight junctions (TJs), adherens junctions (AJs) and desmosomes. The junctional structures are typically aligned from the apical to basal sides, while desmosomes are independently distributed in other areas.
According to ultrastructural analysis, AJs were originally defined as closely apposed plasma membrane domains fortified with dense cytoplasmic plaques, lined with actin filament (F-actin) bundles (see M. G. Farquhar and G. E. Palade, “Junctional complexes in various epithelia.” J. Cell Biol. 1963, 17: 375-412). Molecular analysis showed that AJs are cell-cell adhesion sites assembled with actin-based cytoskeleton and several cytoplasmic components wherein typical cadherins function as cell adhesion molecules (see E. Provost and D. L. Rimm, “Controversies at the cytoplasmic face of the cadherin-based adhesion complex.” Curr. Opin. Cell Biol. 1999, 11: 567-572; A. Nagafuchi, “Molecular architecture of adherens junctions.” Curr. Opin. Cell Biol. 2001, 13: 600-603). Similar to other typical cadherins, E-cadherin is a single-pass transmembrane protein whose extracellular domain mediates homophilic recognition and adherens junction in a Ca2+-dependent manner (see M. Takeichi, “Morphogenetic roles of classic cadherins.” Curr. Opin. Cell Biol. 1995, 7: 619-627). E-cadherin associates with actin cytoskeleton through peripheral membrane proteins, including α-catenins, β-catenins, γ-catenins, α-actin and vinculin (see M. Ozawa et al., “The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.” EMBO J. 1989, 8: 1711-1717; A. Nagafuchi et al., “The 102 kd cadherin-associated protein: similarity to vinculin and posttranscriptional regulation of expression.” Cell 1991, 65: 849-857; M. Watabe-Uchida et al., “α-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells.” J. Cell Biol. 1998, 142: 847-857; E. E. Weiss et al., “Vinculin is part of the cadherin-catenin junctional complex: complex formation between alpha-catenin and vinculin.” J. Cell Biol. 1998, 141: 755-764). β-Catenin directly interacts with the cytoplasmic tail of E-cadherin and connects E-cadherin to α-catenin that directly binds to F-actin (see D. L. Rimm et al., “Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex.” Proc. Nat. Acad. Sci. USA. 1995, 92: 8813-8817). α-Actinin and vinculin are F-actin-binding proteins that directly bind to α-catenin (see M. Watabe-Uchida et al., “α-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells.” J. Cell Biol. 1998, 142: 847-857; E. E. Weiss et al., “Vinculin is part of the cadherin-catenin junctional complex: complex formation between alpha-catenin and vinculin.” J. Cell Biol. 1998, 141: 755-764; K. A. Knudsen et al., “Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin.” J. Cell Biol. 1995, 130: 67-77).
The association of E-cadherin with the actin cytoskeleton through these peripheral membrane proteins potentiate cell-cell adhesion by E-cadherin (see M. Takeichi, “Cadherin cell adhesion receptors as a morphogenetic regulator.” Science 1991, 251: 1451-1455; Y. Imamura et al., “Functional domains of alpha-catenin required for the strong state of cadherin-based cell adhesion.” J. Cell Biol. 1999, 144: 1311-1322).
The present inventors discovered that another cell-cell adhesion molecule (nectin) and F-actin-binding protein (afadin), which associates with nectin, also localize at AJs (see K. Mandai et al., “Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.” J. Cell Biol. 1997, 139: 517-528; K. Takahashi et al., “Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.” J. Cell Biol. 1999, 145: 539-549). Nectin and afadin strictly localized at AJs and, using ultrastructural analysis, they were defined as closely apposed plasma membrane domains fortified with dense cytoplasmic plaques lined with F-actin bundles (see M. G. Farquhar and G. E. Palade, “Junctional complexes in various epithelia.” J. Cell Biol. 1963, 17: 375-412; K. Mandai et al., “Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.” J. Cell Biol. 1997, 139: 517-528; K. Takahashi et al., “Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.” J. Cell Biol. 1999, 145: 539-549). In contrast, E-cadherin is concentrated at AJs but is more widely distributed from the apical to basal sides of the lateral plasma membranes (see B. M. Gumbiner, “Cell adhesion: the molecular basis of tissue architecture and morphogenesis.” Cell 1996, 84: 345-357; S. Tsukita et al., “Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.” Curr. Opin. Cell Biol. 1992, 4: 834-839). Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule (see K. Takahashi et al., “Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.” J. Cell Biol. 1999, 145: 539-549; J. Aoki et al., “Mouse homolog of poliovirus receptor-related gene 2 product, mPRR2, mediates homophilic cell aggregation.” Exp. Cell Res. 1997, 235: 374-384; M. Lopez et al., “The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule.” Blood 1998, 92: 4602-4611; M. Miyahara et al., “Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction.” J. Biol. Chem. 2000, 275: 613-618; K. Satoh-Horikawa et al., “Nectin-3, a new member of immunoglobulin-like cell adhesion molecules that shows homophilic and heterophilic cell-cell adhesion activities.” J. Biol. Chem. 2000, 275: 10291-10299; N. Reymond et al., “Nectin4/PRR4: A new afadin-associated member of the nectin family that trans-interacts with nectin1/PRR1 through V domain interaction.” J. Biol. Chem. 2001, 276: 43205-43215). At present, nectin comprises a family consisting of four membranes, i.e., nectin-1, -2, -3 and -4. All nectins, with the exception of nectin-4, have two or three splice variants, i.e., nectin-1α, -1β, -2α, -2δ, -3α, -3β and -3γ isoforms (see K. Takahashi et al., “Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.” J. Cell Biol. 1999, 145: 539-549; K. Satoh-Horikawa et al., “Nectin-3, a new member of immunoglobulin-like cell adhesion molecules that shows homophilic and heterophilic cell-cell adhesion activities.” J. Biol. Chem. 2000, 275: 10291-10299; N. Reymond et al., “Nectin4/PRR4: A new afadin-associated member of the nectin family that trans-interacts with nectin1/PRR1 through V domain interaction.” J. Biol. Chem. 2001, 276: 43205-43215; M. E. Morrison and V. R. Racaniello, “Molecular cloning and expression of a murine homolog of the human poliovirus receptor gene.” J. Virol. 1992, 66: 2807-2813; J. Aoki et al., “Amino acid residues on human poliovirus receptor involved in interaction with poliovirus.” J. Biol. Chem. 1994, 269: 8431-8438; F. Eberle et al., “The human PRR2 gene, related to the human poliovirus receptor gene (PVR), is the true homolog of the murine MPH gene.” Gene 1995, 159: 267-272; M. Lopez et al. “Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene.” Gene 1995, 155: 261-265; F. Cocchi et al., “The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D.” Proc. Natl. Acad. Sci. USA. 1998, 95: 15700-15705). Nectin-1 was originally identified as one of the poliovirus receptor-related proteins (PRR1) (see M. Lopez et al., “Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene.” Gene 1995, 155: 261-265). Nectin-2 was originally identified as a murine homolog of human poliovirus receptor protein (see M. E. Morrison and V. R. Racaniello, “Molecular cloning and expression of a murine homolog of the human poliovirus receptor gene.” J. Virol. 1992, 66: 2807-2813), but turned out to be another poliovirus receptor-related protein (PRR2) (see F. Eberle et al., “The human PRR2 gene, related to the human poliovirus receptor gene (PVR), is the true homolog of the murine MPH gene.” Gene 1995, 159: 267-272; M. Lopez et al., “Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene.” Gene 1995, 155: 261-265). PRR1 and PRR2 were later shown to serve as receptors for α-herpesvirus, facilitating their invasion and intercellular spreading, and thus were renamed HveC and HveB, respectively (see F. Cocchi et al., “The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D.” Proc. Natl. Acad. Sci. USA. 1998, 95: 15700-15705; F. Cocchi et al., “Cell-to-cell spread of wild-type herpes simplex virus type 1, but not of syncytial strains, is mediated by the immunoglobulin-like receptors that mediate virion entry, nectin1 (PRR1/HveC/HIgR) and nectin2 (PRR2/HveB).” J. Virol. 2000, 74: 3909-3917; R. J. Geraghty et al., “Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.” Science 1998, 280: 1618-1620; M. S. Warner et al., “A cell surface protein with herpesvirus entry activity (HveB) confers susceptibility to infection by mutants of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus.” Virology 1998, 246: 179-189; M. Lopez et al., “Nectin2α (PRR2α or HveB) and nectin2α are low-efficiency mediators for entry of herpes simplex virus mutants carrying the Leu25Pro substitution in glycoprotein D.” J. Virol. 2000, 74: 1267-1274; T. Sakisaka et al., “Requirement of interaction of nectin-1 alpha/HveC with afadin for efficient cell-cell spread of herpes simplex virus type 1.” J. Virol. 2001, 75: 4734-4743). All members of nectin have an extracellular domain with three immunoglobulin-like loops, a single transmembrane region and a cytoplasmic region. Furthermore, all of them, with the exception of nectin-1β, nectin-3γ and nectin-4, have a conserved motif of 4 amino acid residues (Glu/Ala-X-Tyr-Val) at their carboxy terminus, and this motif binds to the PDZ domain of afadin (see K. Mandai et al., “Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.” J. Cell Biol. 1997, 139: 517-528; K. Takahashi et al., “Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.” J. Cell Biol. 1999, 145: 539-549; K. Satoh-Horikawa et al., “Nectin-3, a new member of immunoglobulin-like cell adhesion molecules that shows homophilic and heterophilic cell-cell adhesion activities.” J. Biol. Chem. 2000, 275: 10291-10299; N. Reymond et al., “Nectin4/PRR4: A new afadin-associated member of the nectin family that trans-interacts with nectin1/PRR1 through V domain interaction.” J. Biol. Chem. 2001, 276: 43205-43215).
Afadin has at least two splice variants, namely, l- and s-afadins (see K. Mandai et al., “Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.” J. Cell Biol. 1997, 139: 517-528). l-Afadin, the larger splice variant, binds to nectin and, through its F-actin binding domain, to F-actin. l-Afadin binds to the side of F-actin but does not crosslink with it to form bundles. l-Afadin has two Ras-associated domains (RA), a forkhead-associated domain (FHA), a dilute (DIL) domain, a PDZ domain, two proline rich domains (PR) and an F-actin-binding PR domain (see FIG. 1A). The DIL domain is found in afadin, dilute (DIL) and V-type myocin, comprising Myo2 and Myo4. However, the function of this domain is still unknown (see C. P. Ponting, “AF-6/cno: neither a kinesin nor a myosin, but a bit of both.” Trends Biochem. Sci. 1995, 20: 265-266). Recent findings that the Myo4 region containing the DIL domain binds to an adaptor protein, She3 (see F. Bohl et al., “She2p, a novel RNA-binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p.” EMBO J. 2000, 19: 5514-5524; R. M. Long et al., “She2p is a novel RNA-binding protein that recruits the Myo4p-She3p complex to ASH1 mRNA.” EMBO J. 2000, 19: 6592-6601) indicates that the DIL domain is involved in protein-protein interactions.
s-Afadin, the smaller splice variant, has two RA, FHA, DIL, PDZ and two PR domains, but lacks the F-actin-binding PR domain. Human s-afadin is identical to the gene product of AF-6, a gene that has been identified as an ALL-1 fusion partner involved in acute myeloid leukemias (see R. Prasad et al., “Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation.” Cancer Res. 1993, 53: 5624-5628). Unless otherwise specified, “afadin” refers to l-afadin in the present specification.
Nectin supplies E-cadherin-β-catenin complex to the nectin-based cell-cell adhesion sites through afadin and α-catenin in fibroblast and epithelial cells (see K. Tachibana et al., “Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins.” J. Cell Biol. 2000, 150: 1161-1176; A. Fukuhara et al., “Involvement of Nectin in the Localization of Junctional Adhesion Molecule at Tight Junctions.” Oncogene 2002, 21: 7642-7655). Furthermore, nectin supplies TJ components, including ZO-1, claudin, occludin and junction adhesion molecule (JAM), to the nectin-based cell-cell adhesion sites through afadin in fibroblasts and epithelial cells (see A. Fukuhara et al., “Involvement of Nectin in the Localization of Junctional Adhesion Molecule at Tight Junctions.” Oncogene 2002, 21: 7624-7655; S. Yokoyama et al., “alpha-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin.” Mol. Biol. Cell 2001, 12: 1595-1609; A. Fukuhara et al., “Role of Nectin in Organization of Tight Junctions in Epithelial Cells. Genes Cells.” Genes Cells 2002, 7: 1059-1072). Claudin is an important cell-cell adhesion molecule that forms TJ strands (see M. Furuse et al., “A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts.” J. Cell Biol. 1998, 143: 391-401; M. Furuse et al., “Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.” J. Cell Biol. 1998, 141: 1539-1550; S. Tsukita and M. Furuse, “Occludin and claudins in tight-junction strands: leading or supporting players?” Trends Cell Biol. 1999, 9: 268-273; S. Tsukita et al., “Structural and signalling molecules come together at tight junctions.” Curr. Opin. Cell Biol. 1999, 11: 628-633), and occludin and JAM are other transmembrane proteins at TJs (see S. Tsukita and M. Furuse, “Occludin and claudins in tight-junction strands: leading or supporting players?” Trends Cell Biol. 1999, 9: 268-273; S. Tsukita et al., “Structural and signalling molecules come together at tight junctions.” Curr. Opin. Cell Biol. 1999, 11: 628-633; I. Martin-Padura et al., “Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration.” J. Cell Biol. 1998, 142: 117-127) Claudin, occludin and JAM interact with an F-actin-binding scaffold molecule, ZO-1 (see B. R. Stevenson et al., “Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.” J. Cell Biol. 1986, 103: 755-766; M. Itoh et al., “The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.” J. Cell Biol. 1993, 121: 491-502; M. Itoh et al., “Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments.” J. Cell Biol. 1997, 138: 181-192; M. Itoh et al., “Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin.” J. Biol. Chem. 1999, 274: 5981-5986; M. Itoh et al., “Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.” J. Cell Biol. 1999, 147: 1351-1363; E. Willott et al., “The tight junction protein ZO-1 is homologous to the Drosophila discs—large tumor suppressor protein of septate junctions.” Proc. Natl. Acad. Sci. USA. 1993, 90: 7834-7838; M. Furuse et al., “Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.” J. Cell Biol. 1994, 127: 1617-1626; J. Haskins et al., “ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin.” J. Cell Biol. 1998, 141: 199-208; G. Bazzoni et al., “Interaction of Functional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin.” J. Biol. Chem. 2000, 275: 20520-20526; K. Ebnet et al., “Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1.” J. Biol. Chem. 2000, 275: 27979-27988; E. S. Wittchen et al., “Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly.” J. Cell Biol. 2000, 151: 825-836; M. Itoh et al., “Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions.” J. Cell Biol. 2001, 154: 491-497). In epithelial cells of afadin (−/−) mice and (−/−) embryoid bodies, the proper organization of AJs and TJs is impaired (see W. Ikeda et al., “Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.” J. Cell Biol. 1999, 146: 1117-1132). By positional cloning, nectin-1 has recently been related to cleft lip/palate-ectodermal dysplasia, which is characterized by cleft lip/palate, syndactyly, mental retardation and ectodermal dysplasia (see K. Suzuki et al., “Mutations of PVRL1, encoding a cell-cell adhesion molecule/herpesvirus receptor, in cleft lip/palate-ectodermal dysplasia.” Nat. Genet. 2000, 25: 427-430).
In addition, the present inventors have recently identified that the nectin-afadin system is involved in the formation of synapses of neurons in cooperation with N-cadherin (see A. Mizoguchi et al., “Nectin: an adhesion molecule involved in formation of synapses.” J. Cell Biol. 2002, 156: 555-565) and that the nectin-afadin system constitutes an important adhesion system in the organization of. Sertoli cell-spermatid junction of the testis (see K. Ozaki-Kuroda et al., “Nectin couples cell-cell adhesion and the actin scaffold at heterotypic testicular junctions.” Curr. Biol. 2002, 12: 1145-1150). Therefore, nectin and afadin are important for the formation of a wide variety of intercellular junctions either together with or independently of known cell adhesion molecules. However, the molecular mechanism how the nectin-afadin system organizes these intercellular junctions is not yet fully understood.